The present invention relates generally to the field of plant breeding. More specifically, it relates to gene identification in plants.
The ability to predict the inheritance of certain traits is of tremendous value to agricultural, horticultural, and medical endeavors. For traits controlled by single genes, predicting inheritance patterns is often no more difficult than understanding simple Mendelian principles. However, traits controlled by more than one locus offer unique challenges. Statistical methods and experimental designs have been created in an attempt to predict the inheritance of numerous quantitatively inherited phenotypic traits.
However, attempts to compare gene expression between groups of organisms separated on the basis of phenotype of complexly inherited traits have still been frustrating because phenotypes are the result of environmental factors plus the effects of many genes. If a trait is complexly inherited, no individual in a segregating population is expected to carry all favorable or unfavorable alleles. Therefore, each group consists of expression products of both favorable and unfavorable alleles at loci affecting the trait.
What is needed in the art is a method to associate a gene or an expression product with a phenotypic trait of interest for use in such applications as predicting the inheritance of quantitatively inherited phenotypic traits and in separating groups of organisms on the basis of allelic variation rather than solely on phenotypic variation. The present invention provides these and other advantages.
Generally, it is the object of the present invention to provide methods of selection of a gene associated with a phenotypic trait. It is an object of the present invention to provide a method of associating a gene with a phenotypic trait of interest and methods of associating an expression product with a phenotypic trait of interest.
Therefore, in one aspect, the present invention relates to a method of associating a gene with a phenotypic trait of interest comprising (a) segregating members of a biological population by the presence or absence of one or more genetic markers statistically associated with a quantitatively inherited phenotypic trait; (b) expression profiling segregated members of (a); and, (c) determining from expression profiles of (b) the gene associated with said phenotypic trait.
In another aspect, the present invention relates to a method of associating an expression product with a phenotypic trait of interest comprising (a) segregating members of a population consisting of a biological population by the presence or absence of one or more genetic markers statistically associated with said phenotypic trait, wherein said phenotypic trait has a statistical association with more than one genetic locus; (b) expression profiling at least one segregated member of (a) possessing said genetic marker and at least one segregated member of (a) lacking said genetic marker; and, (c) determining from said expression profiles of (b) an expression product associated with said phenotypic trait.
In yet another aspect, the present invention relates to associating an expression product with a phenotypic trait of interest, comprising: (a) expression profiling a plurality of members of a biological population having one or more genetic markers statistically associated with a phenotypic trait of interest wherein said phenotypic trait exhibits statistical association with more than one genomic locus; (b) expression profiling a plurality of members from said population lacking said genetic marker; and, (c) determining from expression profiles of (a) and (b) an expression product associated with said phenotypic trait.
Definitions
The terms defined below are more fully defined by reference to the specification as a whole. Units, prefixes, and symbols may be denoted in their SI accepted form. Numeric ranges are inclusive of the numbers defining the range and include each integer within the defined range.
The phrase xe2x80x9cbiological populationxe2x80x9d includes reference to a group of individuals having the capacity to be genetically crossed, regardless of species. For example, a group of Glycine soja and Glycine max plants would be considered a xe2x80x9cbiological populationxe2x80x9d because they are capable of being crossed. Individuals, as used herein, will refer to whole organisms, organism organs, cells, and progeny of same. For example, a plant biological population would include reference to whole plants, plant organs, plant cells, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.
The phrase xe2x80x9cexpression profilingxe2x80x9d includes reference to generating an expression profile. By xe2x80x9cexpression profilexe2x80x9d is meant the quantitation of a plurality of DNA, RNA, or protein expression products from a cell, tissue or whole organism. Many RNA expression products of a cell or tissue can simultaneously be detected on a nucleic acid array, or by the technique of differential display or modification thereof, such as those described in WO 97/15690 by Rothberg et al. and U.S. Pat. No. 5,719,060.
By xe2x80x9cgenetic locusxe2x80x9d is meant a location on a chromosome. By xe2x80x9cgenomic locusxe2x80x9d is meant a location within the entire set of chromosomes of an organism.
As used herein, xe2x80x9clinkage disequilibriumxe2x80x9d refers to a statistical association between two loci or between a trait and a marker.
As used herein, xe2x80x9cmarkerxe2x80x9d includes reference to a locus on a chromosome that serves to identify a unique position on the chromosome. A genotype may be defined by use of one or a plurality of markers.
xe2x80x9cPhenotypic traitsxe2x80x9d may be comprised of, but are not limited to, a combination of measurable traits reflected in, but not limited to, the following:
Barren plants: The percent of plants per plot that were barren (lack ears).
Brittle Stalks: This is a measure of the stalk breakage near the time of pollination, and is an indication of whether the stalk of a hybrid or inbred would snap or break near the time of flowering under severe winds.
Yield: Yield of the grain at harvest in bushels per acre adjusted to 15.5% moisture.
Disease resistance: Resistance to any plant pathogen or group of plant pathogens.
Drydown: The relative rate at which a hybrid will reach acceptable harvest moisture compared to other hybrids.
Dropped Ears: A measure of the number of dropped ears per plot and represents the percentage of plants that dropped ears prior to harvest.
Ear height: Ear height is a measure from the ground to the highest placed developed ear node attachment and is measured in inches.
General Ear Mold: This is based on overall rating for ear mold of mature ears without determining the specific mold organism, and may not be predictive for a specific ear mold.
European Corn Borer feeding resistance (Ostrinia nubilalis): Average inches of tunneling per plant in the stalk or post flowering degree of stalk breakage and other evidence of feeding by European Corn Borer.
European Corn Borer Dropped Ears (Ostrinia nubilalis): Dropped ears due to European Corn Borer. Percentage of plants that dropped ears under second generation corn borer infestation.
Early Growth: scored when two leaf collars are visible.
Early Stand Count: This is a measure of the stand establishment in the spring and represents the number of plants that emerge on per plot basis for the inbred or hybrid.
Growing Degree Units: Using the Barger Heat Unit Theory, that assumes that maize growth occurs in the temperature range 50xc2x0 F.-86xc2x0 F. and that temperatures outside this range slow down growth; the maximum daily heat unit accumulation is 36 and the minimum daily heat unit accumulation is 0. The seasonal accumulation of GDU is a major factor in determining maturity zones.
GDU to physical maturity: The number of growing degree units required for an inbred or hybrid line to have approximately 50 percent of plants at physiological maturity from time of planting. Growing degree units are calculated by the Barger method (described below).
GDU to shed: The number of growing degree units (GDUs) or heat units required for an inbred line or hybrid to have approximately 50 percent of the plants shedding pollen and is measured from the time of planting. Growing degree units are calculated by the Barger Method, where the heat units for a 24-hour period are:   GDU  =                    (                  Max          .                      xe2x80x83                    ⁢          temp          .                      xe2x80x83                    ⁢                      +                          xe2x80x83                        ⁢                          Min              .                              xe2x80x83                            ⁢              temp              .                                      )            2        -    50  
The highest maximum temperature used is 86xc2x0 F. and the lowest minimum temperature used is 50xc2x0 F. For each inbred or hybrid it takes a certain number of GDUs to reach various stages of plant development.
GDU to silk: The number of growing degree units required for an inbred line or hybrid to have approximately 50 percent of the plants with silk emergence from time of planting. Growing degree units are calculated by the Barger Method as given in GDU SHD definition.
Grain Appearance: The general appearance of the shelled grain as it is harvested based on such factors as the color of harvested grain, any mold on the grain, and any cracked grain.
Harvest Moisture: The moisture is the actual percentage moisture of the grain at harvest.
Moisture Advantage: The moisture advantage of variety #1 over variety #2 as calculated by: Moisture of variety #2-Moisture of variety #1=Moisture Advantage of variety #1.
Grain Oil: The amount of the kernel that is oil, expressed as a percentage on a dry weight basis.
Plant Height: This is a measure of the height of the plant from the ground to the tip of the tassel in inches.
Pollen Score: Rating indicating the amount of pollen shed.
Pollen Weight: This is calculated by dry weight of tassels collected as shedding commences minus dry weight from similar tassels harvested after shedding is complete. Predicted Relative Maturity (PRM). This trait, predicted relative maturity, is based on the harvest moisture of the grain. The relative maturity rating is based on a known set of checks and utilizes standard linear regression analyses and is referred to as the Comparative Relative Maturity Rating System that is similar to the Minnesota Relative Maturity Rating System.
PRM Shed: Predicted relative maturity based on shed is based on the growing degree units (GDU) required to reach 50% pollen shed. Relative values are predicted values from the linear regression of observed GDU""s on relative maturity of commercial checks.
Protein Rating: Comparison of relative amounts of protein in the grain compared to hybrids of similar maturity.
Root lodging: The percentage of plants that root lodge; plants that lean from the vertical axis at an approximately 30xc2x0 angle or greater would be counted as root lodged.
Scatter Grain: lack of pollination or kernel abortion on the ear.
Seedling Vigor: The amount of vegetative growth after emergence at the seedling stage (approximately five leaves for maize).
Stay green: the measure of plant health near the time of black layer formation (physiological maturity).
Stand (%): the percent of stalks standing at harvest.
Stalk Count: the final stand or number of plants per plot.
Stalk Lodge: the percentage of plants that stalk lodged (stalk breakage) as measured by either natural lodging or pushing the stalks and determining the percentage of plants that break below the ear.
Tassel Blast: the degree of blasting (necrosis due to heat stress) of the tassel at the time of flowering.
Tassel Size: the relative size of the tassel.
Tassel Weight: this is the average weight of a tassel (grams) just prior to pollen shed.
Ear Texture: the relative hardness (smoothness of crown) of mature grain.
Number of tillers: a count of the number of tillers per plot that could possibly shed pollen.
ASI: the interval in GDU""s between the GDU to shed and GDU to silk.
Grain composition amino acids: The average amount and type of amino acids present in the kernel based on 25 kernels.
Grain composition carbohydrate: The average amount and type of carbohydrate present in the kernel based on 25 kernels.
Ear length: The length of the ear from the base to the tip of the cob.
Kernel Row Count: The number of rows of kernels per ear.
Kernel per row: The average number of kernels per row based on at least 4 rows.
Ear diameter: The average diameter of the ear with intact kernels based on three measurements at different places on the ear.
Kernel row length: The average distance from the first kernel at the base of the ear to the last kernel at the tip of the ear.
KWT100: The average mass of kernel in grams for 100 kernels either as fresh tissue or dried to moisture level of 15.5%.
KWT300: The average mass of kernel in grams for 300 kernels either as fresh tissue or dried to moisture level of 15.5%.
The term xe2x80x9cstatistically associatedxe2x80x9d refers to the tendency of two events to occur together at a frequency greater than that attributable to chance, where the frequency attributable to chance is represented by a pre-determined level of significance. Statistical association can be determined by any one of a number of significance tests well known to those in the art, for example, ANOVA or t-tests. See, e.g. Statistical Methods, Snedecor, G. W. and Cochran, W. G., Iowa State University Press, Ames, Iowa (1985). Significance levels for xcex1 are preferably less than 0.01. For example, levels of significance for this invention could range between 0 and about 0.250, e.g. less than about 0.0001, 0.00050, 0.0010, 0.0050, 0.010, 0.025, 0.050, 0.100, or 0.250.